Cell identification with nanoparticles, compositions and methods related thereto

Abstract

The present disclosure provides a method for identifying rare or low-abundant biological entities such as Hodgkin's and Reed-Sternberg cells, circulating tumor cells in peripheral blood, circulating fetal cells, stem cells, somatic cells, HIV-infected T cells, bacteria or viruses in water, adenoviruses, enteroviruses, hepatitis A and E, dengue, Swine Flu, bovine diarrhea, and protozpa/helminthes. The method uses a suite of nanoparticle-conjugated agents to mark biological targets of interest for subsequent fluorescence imaging. In certain embodiments, the nanoparticle-conjugated agents are fluorescent semiconductor nanocrystals conjugated with antibodies with affinity for CD15, CD30, CD45, and Pax5. In certain embodiments, a method is developed to differentiate Hodgkin's and Reed-Sternberg (HRS) cells from amongst surrounding immune cells such as T and B lymphocytes with greater specificity and precision than traditional immunohistochemistry (IHC) for the diagnosis of Hodgkin's lymphoma.

We claim: 
     
         1 . A method comprising mixing a sample with a nanoparticle-conjugated marker with affinity for a target providing a marked target wherein said target is a primary antibody with affinity to a cell biomarker. 
     
     
         2 . The method of  claim 1 , wherein the cell biomarker is a cell surface biomolecule or an intracellular biomolecule. 
     
     
         3 . The method of  claim 1 , wherein said sample comprises a human lymphoma tissue that has been incubated with at least one primary antibody each primary antibody having an epitope to a fragment of a protein selected from the group consisting of CD15, CD30, CD45, and Pax5. 
     
     
         4 . A method of  claim 3  further comprising the step of mixing the human lymphoma tissue with a second set of primary antibodies having epitopes selected from the group consisting of CD15, CD30, CD45, and Pax5 that were not used in the first instance wherein the nanoparticle-conjugated marker is a mixture of nanoparticle-conjugated secondary antibodies each with affinity to a different primary antibody used in the second instance. 
     
     
         5 . A method of  claim 4  further comprising the step of exposing the human lymphoma tissue to light; and diagnosing Hodgkin's Lymphoma by identifying a distinct emission pattern for the nanoparticle-conjugated secondary antibodies identifying Hodgkin's and Reed-Sternberg cells 
     
     
         6 . A kit for diagnosing Hodgkin's lymphoma in a subject, the kit comprising: primary antibodies with epitopes for CD15, CD30, CD45, and Pax5; and a plurality of quantum dot-conjugated secondary antibodies to the primary antibodies. 
     
     
         7 . The kit of  claim 6 , wherein the quantum dot-conjugated secondary antibodies are water soluble. 
     
     
         8 . A method of diagnosing benign lymphoid hyperplasia, the method comprising:
 performing the method of  claim 5 ; and identifying benign cells by the absence of the distinct emission pattern for Hodgkin's and Reed-Sternberg cells.   
     
     
         9 . The method of  claim 1 , further comprising imaging said sample with a multispectral imaging system. 
     
     
         10 . A method of detecting a cell in a tissue sample comprising
 a. mixing the sample with a first primary antibody with an epitope to a first cell biomarker and a second primary antibody with an epitope to a second biomarker providing antibody conjugated cells comprising the first and second cell biomarkers;   b. mixing the antibody conjugated cells with a first secondary antibody conjugated to a first nanoparticle with an epitope to the first primary antibody and a second secondary antibody conjugated to a second nanoparticle with an epitope to the second primary antibody providing antibody conjugated cells marked with first and second nanoparticles, provided that the first and second nanoparticles fluoresce at detectably different wavelengths;   c. exposing the sample with antibody conjugated cells marked with first and second nanoparticles to light;   d. detecting a fluorescence pattern for a cell indicative of the first and second cell biomarkers.   
     
     
         11 . The method of  claim 10 , further comprising the step of e) assigning a first pattern of fluorescence to a normal cell; and f) correlating a second pattern of fluorescence to an irregular cell. 
     
     
         12 . The method of  claim 11 , further comprising the step of g) reporting the presence of an irregular cell to the subject. 
     
     
         13 . The method of  claim 10 , wherein the tissue sample is human lymphoma tissue. 
     
     
         14 . The method of  claim 11 , wherein the normal cell is a T and B lymphocyte and the irregular cell is a Hodgkins' and Reed-Sternberg cell. 
     
     
         15 . The method of  claim 10 , wherein the first cell biomarker is selected from CD15, CD30, CD45, and Pax5 and the second cell biomarker is selected from CD15, CD30, CD45, and Pax5 provided it is not the same as the first biomarker. 
     
     
         16 . A method of detecting a cell in a tissue sample comprising
 a. mixing the tissue sample with an antibody-conjugated nanoparticle each having an epitope to a target biomolecule, providing an antibody conjugated cell marked with an antibody-conjugated nanoparticle, provided that two or more antibody-conjugated nanoparticles are used that fluoresce at detectably different wavelengths from each other and bind different target biomolecules;   b. exposing the tissue sample with antibody conjugated cells marked with a nanoparticle to light;   c. detecting a fluorescence pattern for a targeted cell.   
     
     
         17 . The method of  claim 16 , further comprising the step of e) assigning a first pattern of fluorescence to a normal cell; and f) correlating a second pattern of fluorescence to an irregular cell. 
     
     
         18 . A method of detecting a cell in a tissue sample comprising
 a. mixing the sample with a first marker that binds a cell biomarker providing marker conjugated cells;   b. mixing the marker conjugated cells with a secondary marker conjugated to a nanoparticle that binds to the first marker providing marker conjugated cells marked with nanoparticles;   c. exposing the sample with marker conjugated cells marked with nanoparticles to light;   d. detecting a fluorescence pattern for a cell indicative of the cell biomarker.   
     
     
         19 . The method of  claim 17  wherein the tissue sample is lymphoma tissue. 
     
     
         20 . The method of  claim 17 , wherein steps a and b are repeated two or more times with first markers that bind different cell biomarkers.